In the current Ebola virus (EBOV) epidemic, the laying of hands on the dead serves a clear mode of transmission, suggesting that infectious virus is present on the skin of EBOV-infected individuals at death. While significant evidence exists that EBOV can be readily transmitted from the skin of infected individuals late during infection, almost nothing is known about which skin cells support EBOV infection and how the virus passes to the skin's surface. For high titer virus to be present on the skin, it is likely tht skin cells must be infected, productively replicate, and disseminate EBOV. Skin is a complex organ consisting of a variety of epidermal, dermal, and hypodermal cell types that could be susceptible to EBOV infection, including keratinocytes, dermal fibroblasts, sebaceous gland cells, sweat gland cells, endothelial cells, melanocytes, Langerhans cells, and subcutaneous adipocytes. To readily pass to the surface of intact skin, the virus would need to either infect th epidermal keratinocytes directly or pass through the epidermis through sebaceous or sweat gland secretion. Yet, it is unknown whether cells that compose the glands or epidermal keratinocytes are permissive for EBOV. We hypothesize that at late times during EBOV infection, subsurface cells such as macrophages and endothelial cells may transmit virus to either keratinocytes or glands where they support EBOV replication and are involved in producing EBOV found on the surface of the skin. In aim 1, we propose studies to define which skin cells support EBOV infection. Our expertise in EBOV entry (Maury Laboratory) and skin cell culture and immortalization (Klingelhutz Laboratory) will be important for the success of this work. Already, in preliminary studies using a BSL2 model of EBOV infection, we have shown that human epidermal keratinocytes are susceptible to EBOV infection and spread. We will further characterize which other skin cell types can be infected by EBOV in monolayers, 3D organotypic cultures and human skin explants. Collaborations are also in place to determine which skin cells are infected by EBOV in macaques. Studies in aim 2 will determine which surface receptors are used by EBOV in the skin cells. Understanding how virus is passed to the skin's surface is likely to provide impetus to improve safety practices used by health care workers and alter burial practices to prevent spread. Importantly, our studies will point to new avenues for the development of anti-infective agents and anti-viral therapies.